Retrovirus-mediated gene transfer of a globin gene derivative with anti- sickling properties into hematopoietic sem cells (HSCs) remains one of the most promising approaches for the gene therapy of sickle cell anemia We have recently demonstrated that ex-vivo pre-selection of HSCs transduced with a retrovirus containing the human beta-globin gene cis- linked to fragments of the Locus Control Region (LCR) and a Green Fluorescent Protein expression cassette results in complete hematopoietic reconstitution of all transplanted mice with transduced cells and long- term expression of human beta-globin RNA and protein in red blood cells. It is now clear that these phenomena are the direct consequence of the inability of most LCR derivatives to confer position-independent expression on a cis-linked gene integrated at single copy. Accordingly, Specific Aims 1 and 2 will investigate whether or beta- or gamma- globin/LCR retroviruses can be modified to prevent gene silencing. Based on recent advances in our understanding of chromatin-mediated transcriptional possible approaches will include: (i) utilization of chromatin insulators to flank the transferred beta-globin gene and (ii) site- directed mutagenesis of critical CpG dinucleotides in the globin/LCR DNA insert to make it undetectable by the methylation machinery. These non-expressible globin/LCR DNA insert to make it undetectable by the methylation machinery. These non-repressible globin/LCR retroviral constructs will be evaluated concurrently in single proviral copy mouse transgenics and in bone marrow transplantation experiments. Specific Aim 3 will strive to enhance our ability to achieve high-titer viral production of such complex genetic structures without rearrangement. This requires the exclusive production of full-length (unspliced) viral RNA and its efficient packaging. Two approaches will be evaluated. This requires the exclusive production of full-length (unspliced) viral RNA and its efficient packaging. Two approaches will be evaluated: (i) incorporation of a primary transcript stabilizers from the Wood Chuck Hepatitis B virus combined with HIV-1 Rev/RRE components, the most efficient nucleo-cytoplasmic export machinery characterized to date, and (ii) use of an RNA vector derived from the Semliki Forest virus (SFV), whose RNA replicates exclusively in the cytoplasm, to deliver the retroviral RNA into packaging cells and thereby eliminate its unwanted splicing in the nucleus prior to packaging. Finally, these strategies for optimizing both the structure of the globin/LCR construct and its delivery in high titer virus will be adapted in Specific Aim 4 to HIV-1- derived lentiviral vectors for efficient and rapid ex-vivo transduction of quiescent HSC.